photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria

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http://dx.doi.org/10.1039/c2ee22675h

http://pubs.rsc.org/en/journals/journal/EE

Photosynthetic production of ethanol from carbon dioxide in

genetically engineered cyanobacteria

A consolidated bioprocessing strategy is applied to integrate photosynthetic biomass

production and microbial conversion producing ethanol together into the

cyanobacteria.

Page 1 of 35 Energy & Environmental Science

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Photosynthetic production of ethanol from carbon dioxide in

genetically engineered cyanobacteria

Zhengxu Gaoa,b, #

, Hui Zhaoa, #

, Zhimin Lia, Xiaoming Tan

a and Xuefeng Lu*

a

aKey Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics,

Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of

Sciences, No. 189 Songling Road, Qingdao 266101, China

bGraduate School of Chinese Academy of Sciences, Beijing 100049, China

#These authors contributed equally to this work.

*Corresponding author:

Xuefeng Lu,

Phone: 011-86-532-80662712, Fax: 011-86-532-80662712

Email: [email protected]

Page 2 of 35Energy & Environmental Science

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Abstract

Rapidly growing demand for energy and environmental concerns about carbon

dioxide emissions make development of renewable biofuels more and more attractive.

Tremendous academic and industrial efforts have been paid to produce bioethanol,

which is one major type of biofuel. The current production of bioethanol is limited for

commercialization because of issues with food competition (from food-based biomass)

or cost effectiveness (from lignocellulose-based biomass). In this report we applied a

consolidated bioprocessing strategy to integrate photosynthetic biomass production

and microbial conversion producing ethanol together into the photosynthetic

bacterium, Synechocystis sp. PCC6803, which can directly convert carbon dioxide to

ethanol in one single biological system. A Synechocystis sp. PCC6803 mutant strain

with significantly higher ethanol-producing efficiency (5.50 g/L, 212 mg/L/day)

compared to previous research was constructed by genetically introducing pyruvate

decarboxylase from Zymomonas mobilis and overexpressing endogenous alcohol

dehydrogenase through homologous recombination at two different sites of the

chromosome, and disrupting the biosynthetic pathway of poly-β-hydroxybutyrate. In

total, nine alcohol dehydrogenases from different cyanobacterial strains were cloned

and expressed in E. coli to test ethanol-producing efficiency. Effects of different

culturing conditions including tap water, metal ions, and anoxic aeration on ethanol

production were evaluated.


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Introduction

With the rapidly increasing consumption of energy and continuously growing

concerns about climate change,1 renewable biofuels as an alternative energy resource

to the current fossil fuels have attracted more and more attention.2 The production of

bioethanol, which is one major type of biofuel and can be blended with gasoline in

various ratios for use in the unmodified engines, has recently gained tremendous

attention.3, 4

Currently, bioethanol is mainly produced from starch or sugar-rich

agricultural biomass as the feedstock, such as sugarcane in Brazil and corn in US.

Excessive exploiting of food-based feedstock to produce bioethanol would lead to

competition with the world food supply, increase of the food prices, and problems

with food security.5, 6

Another way of bioethanol production is to use non-edible

lignocellulose biomass as feedstock,7, 8

the most abundant form of carbon on the earth.

However, the cost of pretreatment and enzymatic hydrolysis9, 10

and high energy

consumption required11

in the process of producing lignocellulose-based bioethanol

make it less economically competitive.

Thus, there is a significant need to develop innovative technical routes for

production of bioethanol that are not biomass-based but directly

photosynthesis-derived. Photosynthetic bacteria, e.g. cyanobacteria, are potential

candidates as they harbor photosynthetic capability to convert carbon dioxide to

organic carbon metabolites with utilization of solar energy through the Calvin cycle

and can be genetically modified to assemble an ethanol-producing pathway to

metabolically convert organic carbon metabolites to ethanol product (Figure 1). A

theoretical calculation shows that the productivity of ethanol in a photosynthetic

organism can reach ca. 5,280 gal/acre/year.12

In contrast, the annual yield of ethanol

from corn is 321 gal/acre/year, from sugar cane 727 gal/acre/year,13

from switchgrass

330–810 gal/acre/year, and from corn stover 290–580 gal/acre/year.14

Intrinsically

cyanobacteria-based technology for bioethanol production is a consolidated

bioprocessing strategy to integrate photosynthetic biomass production and microbial

conversion producing ethanol together into a bacterium that can directly convert


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carbon dioxide to ethanol in one single biological system, and can avoid using

food-based biomass that causes food supply issues or lignocellulose-based biomass

with low degradation efficiency and high process cost.

In the past couple of years, cyanobacteria have been modified to produce different

types of biofuels and display huge potential for biotechnology applications.15-17

For

instance, cyanobacteria have been genetically engineered to produce ethylene (37

mg/L),18

ethanol (200-500 mg/L)19, 20

and isoprene (0.05 mg/g dry cell/day).21

More

recently, Liao and his coworkers reported the production of isobutyraldehyde (1,100

mg/L),22

isobutanol (450 mg/L)22

and 1-butanol (14.5 mg/L)23

in genetically

engineered Synechococcus elongates PCC7942. Liu et al. described fatty acid

production in genetically modified Synechocystis sp. PCC6803 with the yield of 197

mg/L.24

Our group showed the production of fatty alcohols and alkanes in genetically

engineered Synechocystis sp. PCC6803 previously.25

Furthermore, researchers at LS9

Inc. identified alkane biosynthetic pathway in cyanobacteria, which opens a door for

microbial production of hydrocarbons, major components of current fossil fuels.26

In 1999, Deng and Coleman19

reported the first case of ethanol production by

genetic engineering in Synechococcus sp. PCC7942 which expressed pyruvate

decarboxylase and alcohol dehydrogenase Ⅱ from Zymomonas mobilis under control

of the rbcLS promoter; the amount of ethanol accumulation reached approximately 5

mM (0.23 g/L). Ten years later, Dexter and Fu20

demonstrated that bioethanol can be

produced in Synechocystis sp. PCC6803 with the yield of ca. 10 mM (0.46 g/L).

Algenol Biofuels Inc. recently constructed strains of Synechocystis sp. PCC6803 with

the integration of pyruvate decarboxylase from Z. mobilis and endogenous alcohol

dehydrogenase slr1192 under control of different promoters, with a resulting ethanol

accumulation of 3.6 g/L during 38 days in the culture medium.27

From previously published data, the production yield of ethanol in cyanobacteria is

still far below the theoretical yield (Table 1).12

In an effort to increase the ethanol

productivity in cyanobacteria, here an efficient ethanol-producing mutant strain of

Synechocystis sp. PCC6803 was constructed by genetically introducing exogenous

pyruvate decarboxylase from Z. mobilis and overexpressing endogenous alcohol


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dehydrogenase slr1192 from Synechocystis sp. PCC6803 through homologous

recombination at two different sites of the chromosome, and disrupting the

biosynthetic pathway of poly-β-hydroxybutyrate. The eventual ethanol concentration

and productivity achieved were 5.50 g/L and 212 mg/L/day during 26 days cultivation

and remarkably exceeded previous titers reported (Table 1). At the same time, in order

to reduce the culturing cost for scaling-up, effects of different culturing conditions

including tap water, metal ions and anoxic aeration on ethanol production were

evaluated. Meanwhile, to select alcohol dehydrogenase with higher catalytic

efficiency, nine alcohol dehydrogenases from different cyanobacteria strains were

cloned and co-expressed with pyruvate decarboxylase from Z. mobilis in E. coli to test

ethanol-producing efficiency.


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Broader context

Rapidly growing demand for energy and environmental concerns about carbon dioxide

emissions make development of renewable biofuels more and more attractive.

Tremendous academic and industrial efforts have been paid to produce bioethanol,

which is one major type of biofuel. The current production of bioethanol is limited for

commercialization because of issues with food competition (from food-based biomass)

or cost effectiveness (from lignocellulose-based biomass). Thus, there is a significant

need to develop innovative technical routes for production of bioethanol that are not

biomass-based but direct photosynthesis-derived. Photosynthetic bacteria, e.g.

cyanobacteria, are potential candidates as they harbor photosynthetic capability to

convert carbon dioxide to organic carbon metabolites with utilization of solar energy

through the Calvin cycle and can be genetically modified to assemble an

ethanol-producing pathway to metabolically convert organic carbon metabolites to

ethanol product. A theoretical calculation shows that the productivity of ethanol in a

photosynthetic organism can reach ca. 5,280 gal/acre/year. Previously reported

production yield of ethanol in cyanobacteria is still far below the theoretical yield. Here

we constructed a Synechocystis sp. PCC6803 mutant strain with significantly higher

ethanol-producing efficiency (5.50 g/L, 212 mg/L/day) and evaluated crucial culturing

conditions, which displays great application potential for moving forward solar-ethanol

production.


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Materials and methods

Chemicals and reagents

Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich (USA).

Taq DNA polymerase and all restriction enzyme were purchased from Fermentas

(Canada) or Takara (Japan). The kits used for molecular cloning were from Omega

(USA) or Takara (Japan). Oligonucleotides were synthesized and DNA sequencing

was performed by Sunnybio (Shanghai, China).

Strains and plasmids construction

Strains used and constructed in this study were shown in Table S1. E. coli strain

DH5α was used for molecular cloning and E. coli strain BL21 (DE3) was the host for

protein expression. Strain Syn-LY225

was used for control strain. Strain Syn-XT43

was constructed by recombination of plasmid pXT43 into slr0168 site of wild type

Synechocystis sp. PCC6803. Strain Syn-ZG25 was constructed by recombination of

plasmid pZG25 into slr0168 site of wild type Synechocystis sp. PCC6803. Strain

Syn-HZ23 was constructed by recombination of plasmid pHZ23 into slr1993 and

slr1994 (slr9394 for short) sites of wild type Synechocystis sp. PCC6803. Syn-HZ24

was constructed by recombination of plasmid pHZ23 into slr9394 site of Syn-ZG25.

Plasmids used and constructed in this study were shown in Table S1. Plasmid

pZG25 was constructed by insertion of pdc and slr1192 into pFQ20,25

under control

of Prbc promoter. The pdc ORF was amplified and fused in-frame with 6×histidine tail

by PCR with primers pdcF and pdcR using the genomic DNA of Z. mobilis as the

template. The slr1192 fragment was amplified and fused in-frame with 6×histidine tail

by PCR with primers 1192F and 1192R using the genomic DNA of Synechocystis sp.

PCC6803 as the template. The sequences of primers used in this study were shown in

Table S2.

Plasmid pHZ23 was constructed by insertion of the fragment of pdc and slr1192

into pHZ22 which had the up and down homologous recombination arms of slr9394,

encoding the key enzymes in the synthesis of PHB. The up and down homologous

arms of slr9394 cloned into pMD18-T (Takara) were separately amplified with the


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primers 93F and 93R, 94F and 94R using the genomic DNA of Synechocystis sp.

PCC6803 as the template. The pdc and slr1192 expressed cassette was amplified with

primers dc92F and dc92R using the plasmid pZG25 as the template. Plasmid pXT43

was constructed by insertion of pdc and adhⅡ into pFQ20,25

under control of Prbc

promoter. The adhⅡ fragment was amplified and fused in-frame with 6×histidine tail

by PCR with primers adhF and adhR using the genomic DNA of Z. mobilis as the

template.

Plasmid pZG62 was constructed by insertion of pdc into pMSD1528, 29

under

control of T7 promoter. Plasmid pXT113A, pZG35, pZG36, pZG37, pZG38, pZG39,

pZG40, pZG41 and pZG42 were constructed by insertion of slr1192,

Synpcc7942_0459, all0879, alr0895, alr0897, slr0942, sll0990, all2810 and all5334

into pET-28b (Novagen, Germany), respectively. The gene Synpcc7942_0459 was

amplified with primers 0459F and 0459R using the genomic DNA of Synechococcus

sp. PCC7942 as the template. The genes slr0942 and sll0990 were separately

amplified with primers 0942F and 0942R, 0990F and 0990R using the genomic DNA

of Synechocystis sp. PCC6803 as the template. The genes all0879, alr0895, alr0897,

all2810 and all5334 were amplified with primers 0879F and 0879R, 0895F and

0895R, 0897F and 0897R, 2810F and 2810R, 5334F and 5334R using the genomic

DNA of Anabaena sp. PCC 7120 as the template, respectively.

Plasmid pZG63 was constructed by inserting slr1192 and alr0895 into the plasmid

pET-28b. Plasmid pZG64 was constructed through inserting adhⅡ and slr1192 into

the plasmid pET-28b. Plasmid pZG65 was constructed by inserting alr0895 and adh

Ⅱ into the plasmid pET-28b. Plasmid pZG66 was constructed by inserting alr0895,

adhⅡ and slr1192 into the plasmid pET-28b.

All the transformants have been molecularly characterized and identified by the

PCR experiments for proving DNA integration and segregation as shown in

Supplementary Information (Figure S1).

Protein expression and purification

E. coli BL21 (DE3) (Takara) was transformed with plasmid pXT113A, pXT5,

pZG35 and pZG37, respectively. One liter of LB medium was inoculated with 10 mL


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of the overnight cultured transformed E. coli BL21 (DE3) from a single colony and

grown at 37 °C. Cell cultures were induced by adding 0.4 mM

isopropyl-D-thiogalactopyranoside (IPTG) at OD600 = 0.6 and the cultures were

incubated at 16 °C overnight with shaking at 180 rpm. Thereafter, cells were

harvested by centrifugation, resuspended in binding buffer (20 mM Tris-HCl, 50 mM

NaCl and 5 mM imidazole, pH 7.9) and lysed by sonication. The cell lysate was

pelleted by centrifugation at 10,000 g, 4 °C for 30 min. The supernatant was

immediately added to the Ni-NTA resin (Novagen), pre-equilibrated with the binding

buffer, which was gently agitated at 4 °C for 1 h. The resin was transferred into a 5

mL column that was washed sequentially with 5 column volumes of the binding

buffer, five column volumes of the washing buffer containing 20 mM, 60 mM, 100

mM imidazole to remove nonspecifically bound proteins, and then 25 mL of the

washing buffer containing 250 mM imidazole to elute the target protein. The eluted

proteins were examined by using 12% sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). The purified proteins were subsequently desalinated

with 30 mM Tris-HCl (pH 7.9) and concentrated in 30% PEG20000 before

quantitation using the Bradford method. 30

Ethanol production from genetically engineered E. coli

The transformed cells E. coli BL21 (DE3) were grown in 10 mL LB medium at

37 °C overnight. Then 50 µL seed culture was re-inoculated in 50 mL fresh LB

medium without other carbon sources. The cultures were incubated at 37 °C with

shaking at 80 rpm and induced by 0.4 mM IPTG. The OD600 and ethanol production

was detected every two hours during cell growth.

Transformation of Synechocystis sp. PCC6803

Transforming plasmids into Synechocystis sp. PCC6803 was carried out with the

published method.31

Synechocystis sp. PCC6803 cultures were grown to exponential

phase. The cells were collected by centrifugation, washed with fresh liquid BG11

twice, and resuspended to a density of about 1 × 109 cells/mL. Then the cell

suspension was mixed with plasmid DNA to a final concentration of 10 µg/mL. The

cell and plasmid mixture was incubated for 5 h at 30°C under luminous intensity of


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approximately 50 µE m-2

s-1

before spreading on nitrocellulose filters, which were

rested on BG11 agar plates without antibiotic for 24 h. Finally, the filters were moved

to selective BG11 agar plates containing antibiotics with an appropriate concentration.

After 1-2 week incubation, a single colony was separated and grown in liquid BG11

medium for examination.

Extracting crude enzyme of cells

Cyanobacteria culture (300 mL) was centrifuged at OD730 of 2.0. Then the

harvested cells were resuspended in 10 mL Tris buffer (30 mM Tris-HCl, pH 8.0) and

lysed by sonication in ice-water mixture. The cellular extracts were centrifuged at

10,000 g, 4 °C for 30 min. The supernatant was collected for Western blot analysis or

enzymatic assay.

SDS-PAGE and Western blot analysis

The purified or extracted proteins were separated on 12% SDS-PAGE according to

standard procedure and blotted to PVDF membranes, sealed in 5% nonfat milk-TBST

(0.05% Tween-20 in TBS) at 4 °C overnight. Firstly, the membranes were incubated

with the anti-6×His-tag monoclonal antibodies (from mouse, Tiangen, China) for 2 h

and washed three times with TBST (15 min each). Secondly, the membranes were

incubated with an alkaline phosphatase-linked secondary antibody (goat anti mouse,

Tiangen, China) for 1 h and washed three times with TBST (15 min each). Finally, the

membranes were colored using BCIP/NBT in an Alkaline Phosphatase Color

Development Kit (Amresco, USA) following the manufacturer’s instructions.

Ethanol production assay

To measure the ethanol production more accurately, a condenser and a recovery

bottle were connected with the column bioreactor due to the volatility of ethanol

(Figure S4). This method was not used in the previous study.19, 20

One milliliter

sample from the column bioreactor and 500 µL sample from recovery bottle were

collected every two days separately. Each sample of cell culture was centrifuged at

10,000g for 2 minutes, and the supernatant was used for ethanol assay by a SBA-40c

biosensor analyzer (Shandong Academy of Sciences, China) equipped with the

ethanol oxidase immobilized membrane,32

and the recovered ethanol was also


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measured using this method. Finally, we calculated the total ethanol production by

adding the ethanol production in both the bioreactor and the recovery bottle.

Various cultivation conditions of Synechocystis sp. PCC6803

Cultivation of cyanobacteria with flask

The Synechocystis sp. PCC6803 cells were inoculated in 500 mL flask

containing 300 mL BG11 medium with constant 50 µE.m-2

.s-1

white light at the initial

OD730 of 0.05, and pumped with air or 5% CO2-air (V/V) at 30 °C.

Cultivation of cyanobacteria with column photo-bioreactor

The column bioreactor was a 580 mm×30 mm glass column with a rubber plug.

The cells were grown in a flask to the exponential phase and harvested by

centrifugation. The harvested cells were re-suspended in fresh BG11 medium using

tap water or distilled water, and transferred to the column photo-bioreactor at an

appropriate OD730 with constant 100 µE.m-2

.s-1

white light at 30 °C. For normal

cultivation, the cultures were sparged with 5% CO2-air (normal condition). For

cultivation under anoxic conditions, 5% CO2-N2 was pumped into the cultures with

(anoxic condition-2) or without (anoxic condition-1) the addition of 20 µM

3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the medium after 10 d’s

culturing. The rate of gas addition is about 200 mL/min. And for evaluating the

ethanol yield of the third generation ethanol producer, a condensation device (Figure

S4) was specially assembled to the outlet of the column photo-bioreactor in order to

recovering the evaporated ethanol.

Alcohol dehydrogenase activity assay in vitro

The measurement of alcohol dehydrogenase activity was carried out by

monitoring the decrease in absorbance at 340 nm according to the previous study33

with utilization of different cofactor such as NAD(H) or NADP(H), and

Beckman-coulter DU-800 spectrophotometer was used in this progress. The reaction

buffer contained 30 mM Tris (pH 8.0), 200 µM NADP(H), and different

concentrations of acetaldehyde or ethanol.

Pyruvate decarboxylase activity assay in vitro


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The measurement of pyruvate decarboxylase activity was carried out by

monitoring the decrease in absorbance at 340 nm according to the previous study34

with utilization the cofactor of NADPH, and Beckman-coulter DU-800

spectrophotometer was used in this progress. The reaction buffer contained 100 mM

Tris (pH 7.5), 200 µM NADPH, 0.1 mM MgCl2, 0.1 mM thiamine pyrophosphate,

400 µM purified alcohol dehydrogenase (slr1192) and 10 mM pyruvate.

Evaluation of ethanol-producing capability of different alcohol dehydrogenases

in E. coli

The different alcohol dehydrogenases expressing plasmids (pXT113A, pZG35,

pZG36, pZG37, pZG38, pZG39, pZG40, pZG41 and pZG42) were introduced into E.

coli BL21 (DE3) together with pZG62 (harbouring pdc gene driven by PT7 promoter),

respectively. All resulting pdc and adh co-expressing strains as well as pdc expressing

strain of E. coli were cultured in 250 mL flasks containing 100 mL LB medium at the

initial OD730 of 0.4~0.5, and incubated on a rotary shaker with a shaking speed of 200

rpm at 37°C. Expression of the target proteins was induced by adding 0.4 mM

isopropylthiogalactoside (IPTG) before monitoring the ethanol production.

Results and discussions

First generation ethanol producing strain

Based on the previous research, the first generation ethanol producer Syn-XT43 was

constructed by introducing the ethanol-producing pathway of Z. mobilis consisting of

pyruvate decarboxylase and alcohol dehydrogenase II into Synechocystis sp.

PCC6803, which was used instead of Synechococcus sp. PCC7942 as the host strain

used in Deng and Coleman’s work.19

Here, the ribulose-1,5-bisphosphate

carboxylase/oxygenase (Rubisco) promoter Prbc was used instead of the light-driven

psbAII promoter in Dexter and Fu’s report20

to control the expression of pyruvate

decarboxylase and alcohol dehydrogenase in Synechocystis sp. PCC6803. Both the

construction of plasmid pXT43 (Figure 2A) and the transformation of Synechocystis


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sp. PCC6803 host cells with plasmid were carried out according to the published

procedures.25

The genes encoding pyruvate decarboxylase (pdc), alcohol

dehydrogenase II (adh II) and the promoter Prbc were integrated into the neutral

slr0168 site31

of the genome of Synechocystis sp. PCC 6803 (Figure S1 A).

The pdc and adh II genes were successfully expressed in Syn-XT43, which was

confirmed by western blot analysis (Figure 2B). Syn-LY2 without the introduced pdc

and adh II genes was used as a control strain. The time course for cell growth and

ethanol production of Syn-XT43 under different conditions including sparged with air

or 5% mixed CO2-air (V/V) were examined (Figure 2C and 2D). Figure 2C shows

that 5% CO2 increased the rate of cell growth by 2-fold for both Syn-LY2 and

Syn-XT43. Furthermore, at the same culturing condition, Syn-XT43 strain grows to

about half of the cell density level of the control strain Syn-LY2. Fu20

showed that

Synechocystis can tolerate ethanol up to 10.6 g/L in the medium with negligible

impacts on cell growth, hence the ethanol accumulation in the culture of Syn-XT43

(ca. 0.4 g/L, Figure 2D) should have no direct effect on the cell growth. One possible

reason for the lower cell density of Syn-XT43 is that the carbon resource is utilized to

produce ethanol rather than biomass in the case of the control strain without ethanol

production. The other reason might be that the acetaldehyde accumulated in the

medium caused by reverse catalysis of alcohol dehydrogenase II with conversion of

ethanol to acetaldehyde35

is toxic to cells. Algenol biofuels Inc. reported that a certain

amount of acetaldehyde could be detected in Synechocystis sp. PCC6803 mutant

strain harboring the adh II gene from Zymomonas mobilis, which also has a lower

growth rate compared to the control strain.27

The yield of ethanol production in

Syn-XT43 can reach up to 0.4 g/L in flasks with continuously sparged 5% CO2

(four-fold higher than with air, Figure 2D) and is comparable with the previous

results.19, 20

Second generation ethanol producing strain

Syn-ZG25 was generated as the second generation ethanol producer by transformation

of Synechocystis sp. PCC6803 with plasmid pZG25 (Figure 3A and Figure S1 B),

which substituted the adh II gene from Z. mobilis in pXT43 (Figure 2A) with the


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endogenous alcohol dehydrogenase gene (slr1192) of Synechocystis sp. PCC6803.

Western blot analysis showed that the two genes of pdc and slr1192 in Syn-ZG25

were expressed successfully (Figure 3B). Again, the final cell density level of control

strain Syn-LY2 is slightly higher than that of Syn-XT43 and Syn-ZG25 (Figure 3C).

Compared with that of Syn-XT43, the ethanol production capacity of Syn-ZG25

increased by 50% (Figure 3D) to 0.6 g/L. The only difference between Syn-XT43 and

Syn-ZG25 is that the endogenous alcohol dehydrogenase slr1192 was overexpressed

in Syn-ZG25 instead of the exogenous adh II from Z. mobilis in Syn-XT43.

Obviously, this alternation increased the ethanol producing capability. As a result,

Syn-ZG25 was used as the host strain for further genetic modification.

Third generation ethanol producing strain

To further increase the ethanol productivity by cyanobacteria, more genetic

engineering was carried out. Previously, Wu et al.36

constructed a Synechocystis sp.

PCC6803 mutant strain in which an erythromycin resistant cassette substituted the

ADP-glucose pyrophosphorylase gene in the wild-type strain. Experiments showed

that the poly-β-hydroxybutyrate (PHB) content accumulated up to 14% of the dry cell

weight, much higher than that of the wild-type strain (ca. 3.5%) under

photoautotrophic growth condition. This result indicated that the carbon partitioning

in cyanobacteria for PHB production was enhanced by blocking the glycogen

biosynthetic pathway. Therefore, we hypothesize that the flux to pyruvate, the

precursor of ethanol production, may be increased by knocking out related

competitive pathways. PHB was synthesized by many cyanobacteria, including

Spirulina maxima,37

Synechocystis sp. PCC6803,38

which diverts the carbon flux fixed

by Calvin cycle from the ethanol-producing pathway (Figure 1). So, another strain

Syn-HZ23 was constructed by transformation of Synechocystis sp. PCC6803 wild

type with plasmid pHZ23 (Figure 4A), in which the pdc gene from Z. mobilis and

endogenous slr1192 gene were incorporated into the position of genes coding the

enzymes polyhydroxyalkanoate-specific β-ketothiolase (phaA or slr1993 encoding)

and polyhydroxyalkanoate-specific acetoacetyl-CoA reductase (phaB or slr1994

encoding) in the biosynthetic pathway of PHB (Figure S1 C).


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Unexpectedly, the result shows that there is no significant difference in the

production of biomass and ethanol between Syn-HZ23 and Syn-ZG25 (Figure 4B and

4C). Thus, only blocking the biosynthetic pathway of PHB did not increase ethanol

production. Pyruvate is a central metabolite and its physiological concentration is

controlled by many diversified metabolic pathways. The concentration of pyruvate in

Synechocystis cannot be significantly enhanced by only disrupting the synthesis

pathway of PHB.

When Syn-ZG25 was transformed with the plasmid pHZ23 to generate Syn-HZ24,

in which two copies of exogenous pdc gene from Z. mobilis and endogenous slr1192

gene were integrated into Synechocystis sp. PCC6803 at two different sites of the

chromosome, including the neutral slr0168 site and the position of genes phaAB

(Figure S1 D), the ethanol productivity reached 5.50 g/L over 26 days (Figure 4C). In

comparison to the ethanol yield of Syn-ZG25 and Syn-HZ23 over 26 days, this is

4.9-fold higher than that of Syn-ZG25 (1.12 g/L) or 3.7-fold higher than that of

Syn-HZ23 (1.49 g/L). We designated Syn-HZ24 as the third generation ethanol

producer, and surpasses the previous highest concentration of 3.6 g/L during 38 days

claimed by Algenol Biofuels.27

In order to investigate why ethanol production

improved greatly, the pyruvate decarboxylase and alcohol dehydrogenase activities in

crude cell culture extract were measured spectrophotometrically. As shown in Figure

4D, enzymatic activities of both pyruvate decarboxylase and alcohol dehydrogenase

in Syn-HZ24 is approximately two-folds higher than that in Syn-ZG25. This result is

consistent with the protein expression level as shown in Figure 3B by western blot

analysis, in which the protein expression level of Syn-ZG25 is about half that of

Syn-HZ24. Therefore it indicates that higher enzymatic activities of pyruvate

decarboxylase and alcohol dehydrogenase will increase the ethanol productivity. It

will be our next study direction to explore pyruvate decarboxylase and alcohol

dehydrogenase with higher catalytic efficiency or higher expression level.

Enzymatic characterization and evaluation of ethanol-producing capability of

different alcohol dehydrogenases in E. coli


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The only difference between strain Syn-XT43 and Syn-ZG25 is the alcohol

dehydrogenase overexpressed. Specifically, exogenous alcohol dehydrogenase II (adh

II) from Z. mobilis is used in Syn-XT43 and endogenous alcohol dehydrogenase

slr1192 is used in Syn-ZG25. Figure 3D shows that the ethanol yield of Syn-ZG25 is

50% higher than that of Syn-XT43, so the activity of alcohol dehydrogenase plays an

important role in the production of ethanol. Alcohol dehydrogenases exist in many

organisms including cyanobacteria. Based on the annotation in Cyanobase

(http://genome.kazusa.or.jp/cyanobase), nine different alcohol dehydrogenases from

three different cyanobacteria strains were cloned and co-expressed with pyruvate

decarboxylase from Z. mobilis in E. coli (Table S3).

The slr1192 gene in Synechocystis sp. PCC6803 has proved to code a protein

functioning as alcohol dehydrogenase by Vidal et al.33

The cell growth curves (Figure

5A) showed that the cell cannot grow to high density when only pyruvate

decarboxylase was expressed in E. coli due to the toxicity of acetaldehyde generated

by pyruvate decarboxylase. When pyruvate decarboxylase and alcohol dehydrogenase

were co-expressed in E. coli (Figure S3), the cell grows well and there is no

significant difference in both the final cell density and doubling time between strains

with different alcohol dehydrogenases (Figure 5A, 5C and Table S4). The ethanol

production capability of these alcohol dehydrogenases in E. coli was examined

(Figure 5B and 5D). Figure 5B showed that all the E. coli strains with alcohol

dehydrogenase genes can produce ethanol (the one without alcohol dehydrogenase

gene does not generate ethanol). It means that all the selected nine genes possess the

function of alcohol dehydrogenase. The ethanol production ability of slr1192 and

alr0895 are among the top of the selected genes. Furthermore, we examined the

ethanol productivity of E. coli when pdc was co-expressed with different combination

of adh II, slr1192 and alr0895 genes (Figure 5D). Unlike the results shown in Figure

4C, more expressed copies of alcohol dehydrogenase in E. coli did not increase the

ethanol productivity. The strain with only pdc and one copy of slr1192 has the highest

yield.

To further investigate why the ethanol productivity is different when using different


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alcohol dehydrogenases, four alcohol dehydrogenase enzymes were purified (Figure

S2) and characterized (Table 2). The results showed that the activity of slr1192 was

much higher than that of others (up to 74,000-fold difference) when acetaldehyde and

NADPH were used as substrates. Although the activity of adh II was 94-fold higher

than that of slr1192 when acetaldehyde and NADH were used as substrates, the

cellular concentration of NADP(H) is about 10-fold that of NAD(H) in Synechocystis

sp. PCC6803.39

At the same time, all the tested alcohol dehydrogenases prefer

acetaldehyde as substrate other than alcohol as substrate (40-270 fold higher activity

towards acetaldehyde than alcohol, Table 2).

Interestingly, the ethanol productivity of Syn-ZG25 is only about 50% higher than

that of Syn-XT43 (Figure 3D), although the activity of slr1192 in Syn-ZG25 is about

74,000-fold higher than that of adh II in Syn-XT43. This observation indicates that

the pyruvate decarboxylation is probably the rate-limiting step of ethanol production.

It may be promising to explore more pyruvate decarboxylases to test this hypothesis.

Effects of growing Synechocystis mutant in tap water or with addition of

different metal ions on ethanol production

Alcohol dehydrogenase is a metal-dependent enzyme. Metal ions can either inhibit or

increase the enzyme activity.40

The above experiments show that the activity of

alcohol dehydrogenase will affect the ethanol production in Synechocystis sp.

PCC6803. To determine the effect of metal ions on the ethanol production, the effect

of various metal ions on the activity of purified enzyme slr1192 was assayed first

(Figure 6A). The activity of slr1192 without metal ion was arbitrarily defined as

100%. The enzyme activity was 85%inhibited by 10 µM Zn2+

or Co2+

, and only 50%

and 75% activity remained when 50 µM Mn2+

and Cu2+

were used as the metal factors,

respectively. There was no significant effect on the enzyme activity when Mg2+

and

Ca2+

were added to the assay solution.

The concentration of Zn2+

in BG11 medium, the typical culture medium for

cyanobacteria, is 0.77 µM, which can still inhibit 50% activity of slr1192. The

concentrations of Co2+

, Mn2+

, Cu2+

, Mg2+

and Ca2+

in BG11 medium are 0.042 µM,


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9.15 µM, 0.32 µM, 0.30 mM and 0.245 mM, respectively. Therefore, the effect of

different metal ions on the production of ethanol was tested. Five-fold, 1-fold,

1/2-fold and 1/4-fold of commonly used concentrations of metal ions in the BG11

medium were added to the culture. And 1/4-fold concentration of metal ions without

Zn2+

was also examined. Although the activity of the purified slr1192 protein was

significantly inhibited by Zn2+

at even very low concentration, there was no

significant difference in growth and ethanol production of strain Syn-ZG25 with

various concentrations of metal ions (Figure 6B and 6C). Meanwhile we tested the

activity of crude slr1192 protein from the above cultures after 14 days cultivation and

the results suggested that the crude activity was close to each other even with different

concentrations of metal ions in the cell culture (Figure 6D).

Since there is no significant effect of various concentrations of different metal ions

on the ethanol production in Synechocystis, we also tested the ethanol productivity of

Synechocystis sp. PCC6803 cultured with tap water instead of distilled water used in

the aforementioned experiments. Obviously, it is impossible to use distilled water for

future industrialization of ethanol production in cyanobacteria. So it is important to

know if the yield of ethanol production can be affected by using tap water. The results

show that there is no difference for both cell growth (data not shown here) and the

ethanol productivity when the Syn-ZG25 strain was cultivated in tap water and

distilled water (Figure 6E).

Effects of growing Synechocystis mutant under anoxic conditions on ethanol

production

Liao et al.23

demonstrated that oxygen would inhibit the production of 1-butanol in

genetically engineered Synechococcus elongates PCC7942. To test the effect of

oxygen on ethanol production in Synechocystis sp. PCC6803, the Syn-ZG25 strain

was cultivated under normal and anoxic conditions respectively (Figure 7). Unlike the

results reported by Liao,23

comparable ethanol accumulation was obtained under

normal (5% CO2 and 95% air) and anoxic (5% CO2 and 95% N2) conditions during

the first 15 days cultivation (Figure 7B). Interestingly, when pumped with 5% CO2


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and 95% N2, the culture can maintain the ethanol production constantly until 30 days,

whereas the ethanol production drops sharply from 15 days pumped with 5% CO2 and

95% air. It is consistent with the cell densities of corresponding conditions (Figure

7A). At the 10th

day, 20 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) which

can inhibit photosystem Ⅱ’s function was added to the culture medium under the

above anoxic condition. As a result, the cell growth and the ethanol production were

stopped after the addition of DCMU and dramatically lower than that of without

DCMU (Figure 7B).

Future study directions

A robust ethanol-producing cyanobacteria strain with significantly improved titer of

ethanol production has to be genetically constructed for future industrialization of

solar-ethanol production system. Based on the analysis of metabolic network of

ethanol-producing pathway in Synechocystis sp. PCC6803 (Figure 1), it is reasonable

to foresee that the efficiency of ethanol production can be further increased through

genetic engineering. First, metabolic pathways competing for carbon source could be

knocked out or knocked down. These pathways include the biosynthetic pathway of

glycogen, citric acid cycle and acetate metabolic pathway. Second, the enzymes

involved in strengthening pyruvate production could be overexpressed. For example,

overexpressing the RuBisCo enzyme can enhance the carbon fixation efficiency and

overexpression of malate enzyme and pyruvate kinase might increase the metabolic

flux of pyruvate biosynthesis in the Synechocystis cell. And third, the determining

factors for catalytic conversion of pyruvate to ethanol need to be identified and

pyruvate decarboxylase and alcohol dehydrogenase with higher catalytic efficiency

can be screened and applied to increase biosynthetic flux from pyruvate to ethanol

product. Aside from developing excellent ethanol-producing cyanobacteria strains,

other challenges for developing competitively economical large-scale cultivation and

separation systems specifically used for photosynthetic production of evaporated

ethanol product need to be overcome.


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Conclusions

In this study, three generations of genetically engineered Synechocystis sp. PCC6803

strains, Syn-XT43, Syn-ZG25 and Syn-HZ24, with gradually improved ethanol

productivity were constructed. A final ethanol concentration of 5.50 g/L was achieved

over 26 days of cultivation in Syn-HZ24 with genetic introduction of the exogenous

pyruvate decarboxylase from Zymomonas mobilis and endogenous alcohol

dehydrogenase slr1192 into two different sites of the chromosome of Synechocystis sp.

PCC6803 and disruption of the biosynthetic pathway of poly-β-hydroxybutyrate.

Although the enzymatic activity of the purified alcohol dehydrogenase slr1192 protein

can be significantly inhibited by some metal ions (e.g., Zn2+

at 0.77 µM can inhibit

the enzyme activity by 50%), there is no effect on ethanol production in cyanobacteria

and tap water can be used for cultivation of ethanol-producing Synechocystis sp.

PCC6803 mutant strains. Anoxic cultivation conditions can maintain the ethanol

production much longer time without decrease, which is important for saving the cost

of large-scale industrialization.

Acknowledgements

The research was supported by the National High-Tech Research and Development

Program of China (2012AA052103), Qicheng Carbon Energy Inc. and 100-Talent

Program of the Chinese Academy of Sciences (Grant O91001110A). We would like to

thank Dr. Kenneth Reardon for valuable discussion.


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36. G. F. Wu, Z. Y. Shen and Q. Y. Wu, Enzyme Microb Tech, 2002, 30, 710-715.

37. R. De Philippis, A. Ena, M. Guastiini, C. Sili and M. Vincenzini, FEMS Microbiology Letters,

1992, 103, 187-194.

38. G. F. Wu, Q. Y. Wu and Z. Y. Shen, Bioresour Technol, 2001, 76, 85-90.


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Tables

Table 1. The comparison of ethanol production in cyanobacteria from literature and

this study

Table 2. The kinetic characterization of alcohol dehydrogenases with NADP(H) or

NAD(H) as cofactor


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Reference Host strains Ethanol production

Deng and Coleman19

Synechococcus sp. PCC7942 0.23 g/L

Dexter and Fu20

Synechocystis sp. PCC6803 0.46 g/L

Duhring et al.27

Synechocystis sp. PCC6803 3.60 g/L (95 mg/L/Day)

This study Synechocystis sp. PCC6803 5.50 g/L (212 mg/L/Day)

Table 1


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Enzyme Origin

Acetaldehyde + NADPH Ethanol + NADP+

Km

(mM)

kcat

(min-1

)

kcat/Km

(min-1

mM-1)

Km

(mM)

kcat

(min-1

)

kcat/Km

(min-1

mM-1)

slr1192 Synechocystis sp.

PCC6803

1.56

1271

814.7

19.4

268.2

13.81

adhⅡ Zymomonas

mobilis

380

4.22

0.011

146

0.037

2.53×10-4

all0879 Anabaena sp.

PCC 7120

0.13

2.70

20.8

0.65

0.32

0.49

Synpcc7942

_0459

Synechococcus

sp. PCC7942

0.64

1.44

2.25

128

1.08

8.4×10-3

Enzyme Origin

Acetaldehyde + NADH Ethanol + NAD+

Km

(mM)

kcat

(min-1)

kcat/Km

(min-1

mM-1)

Km

(mM)

kcat

(min-1)

kcat/Km

(min-1

mM-1)

slr1192 Synechocystis sp.

PCC6803

9.56

67.7

7.08

730.6

89.3

0.122

adhⅡ Zymomonas

mobilis

2.73

1816

665.2

63.3

581.6

9.19

Table 2


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Figure legends

Figure 1. Pyruvate relevant metabolic pathways in Synechocystis sp. PCC6803. PDC,

pyruvate decarboxylase; ADH, alcohol dehydrogenase; agp, ADP-glucose

pyrophosphorylase; glg, glycogen synthase; pps, phosphoenolpyruvate synthase;

AlaDH, alanine dehydrogenase; ldh, lactate dehydrogenase; phaA, PHA-specific

β-ketothiolase; phaB, PHA-specific acetoacetyl-CoA reductase; Rubisco,

ribulose-1,5-bisphosphate carboxylase/oxygenase; pta, phosphotransacetylase; ackA,

acetate kinase; me, malic enzyme; pyk, pyruvate kinase; acs, acetyl-coenzyme A

synthetase; AldDH, acetaldehyde dehydrogenase

Figure 2. First generation ethanol producer. (A) Plasmid map of pXT43 used to

transform Synechocystis sp. PCC6803. (B) Western blot analysis of protein

expression. Lane 1: the soluble proteins of wild type strain of Synechocystis sp.

PCC6803 was used as negative control. Lane 2: protein marker. Lane 3: the purified

glycerol-3-phosphate dehydrogenase from Thermotoga maritima MSB8 with a

C-terminal 6×histidine tag as positive control (Molecular weight: 37 KDa). Lane 4

and 5: soluble proteins with Histidine tag of Syn-XT43 (two paralleled samples).

Syn-XT43 and Syn-LY2 (control) strains were cultured in flasks pumped with air (Air)

or 5% CO2 (CO2), and growth (C) and ethanol production (D) were measured every

two days.

Figure 3. Second generation ethanol producer. (A) Plasmid map of pZG25 used to

transform Synechocystis sp. PCC6803. (B) Western blot analysis of protein

expression. Lane 1: protein marker, Lane 2: the soluble proteins of wild type strain of

Synechocystis sp. PCC6803 was used as negative control, Lane 3: purified slr1192

with 6xHistidine tag, Lane 4: purified PDC with 6xHistidine tag, Lane 5: soluble

proteins with Histidine tag of Syn-HZ24, Lane 6: soluble proteins with Histidine tag

of Syn-ZG25. Syn-ZG25, Syn-XT43 and Syn-LY2 (control) strains were cultured in

flasks, and growth (C) and ethanol production (D) were measured every two days.


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Figure 4. Third generation ethanol producer. (A) Plasmid map of pHZ23 used to

transform Synechocystis sp. PCC6803 and Syn-ZG25. Syn-ZG24, Syn-ZG23,

Syn-ZG25 and Syn-LY2 (control) strains were cultured on column photobioreactors

with a condensation device, and growth (B) and ethanol production (C) were

measured every two days. (D) The enzymatic activities of pyruvate decarboxylase and

alcohol dehydrogenase in Syn-ZG25 and Syn-HZ24. The activities were measured

with crude cell culture extract at 12 days of cell growth.

Figure 5. Growth curves (A, C) and ethanol production curves (B, D) of E. coli

mutants with heterologous overexpression of pyruvate decarboxylase from

Zymomonas mobilis and different alcohol dehydrogenases.

Figure 6. Effects of growing Synechocystis mutant in tap water or with addition of

different metal ions on ethanol production. (A) The effect of different metal ions on

the activity of pure alcohol dehydrogenase encoded by the slr1192 gene of

Synechocystis sp. PCC6803. (B) Growth curves and (C) Ethanol production curves of

the Syn-ZG25 strain cultivated under column photobioreactor condition in medium

containing various metal ions with different concentrations. (D) Analysis of crude

enzymatic activity of the slr1192 enzyme from the culture of Syn-ZG25 strain

cultivated in BG11 medium containing different ion concentrations. (E) Ethanol

production curves of the Syn-ZG25 strain cultivated in tap water and distilled water.

Figure 7. Effects of growing Synechocystis mutant under anoxic conditions on

ethanol production. Syn-ZG25 strain was cultured on column photobioreactors: (A)

Growth curves. (B) Ethanol production curves. Normal condition: continuously

sparged with 5% CO2 and 95% air. Anoxic condition 1: continuously sparged with 5%

CO2 and 95% N2. Anoxic condition 2: continuously sparged with 5% CO2 and 95%

N2 with addition of 20 µM DCMU after 10 days’ culturing. The blue arrow symbol

indicates the time points when the DCMU was added into the cultures.


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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A B

Figure 7


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photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria

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